Thiol specific antioxidant (TSA) protects glutamine synthetase (GS) from inactivation against the thiol-dependent, metal-catalyzed oxidation (MCO) system, but not by MCO systems in which the thiol compound is replaced by another electron donor (viz. ascorbate). We have previously shown that the protective mechanism of TSA involves H2O2 removal by its intrinsic thiol peroxidase activity: 2 RSH + H2O2 -> RSSR + 2 H2O. In contrast, the thiol peroxidase activity of yeast TSA cannot explain its ability to protect GS from inactivation against MCO systems containing high concentrations of H2O2 (10 mM) (the high H2O2 system). Under these conditions, the rate of H2O2 decomposition in the presence of TSA (0.8 mg/ml) is only slightly higher than in its absence; nevertheless, TSA prevents GS inactivation during a period when H2O2 is still present in substantial amounts. TSA is unable to protect GS against the MCO which comprises ascorbate (10 mM)/Fe(IIl) (3 micromoles)/H2O2 (10 mM)/O2. Other sulfhydryl-containing proteins can also prevent GS inactivation by the high H2O2 system but much higher concentrations are required to achieve a lower level of protection. TSA has two cysteines (47 and 170), which were individually replaced by serine residues (C47S and C170S) (PNAS, 91, 7022-6, 1994). Only C170S can protect GS, indicating that cysteine 47 is required for this peroxidase- independent mechanism of TSA. TSA inhibits oxygen consumption by the high H2O2 system, suggesting that the peroxidase independent mechanism involves the removal of sulfur radical species, since some of these free radicals react very fast with oxygen. TSA can protect GS under argon atmosphere, indicating that the peroxidase-independent activity of TSA does not involve the removal of oxygenated sulfur radicals. Three mammalian proteins homologous to yeast TSA (Mer 5, natural killer enhancer factors A and B) were analyzed for the peroxidase-independent mechanism of TSA. Although all three proteins protect GS when no H2O2 is added, only Mer-5 was able to protect GS against the high H2O2 system.